Commercially-available formats for nucleic acid purification include spin columns, magnetic beads in a tube or the use of vacuum to draw liquids through a column or plate. In these formats, nucleic acids are isolated as follows. The cells are grown in a suitable medium, the culture is centrifuged to collect the cells and the growth medium is discarded. Next, the cells are lysed to release the nucleic acids. Usually, a second centrifugation step is performed after lysis to pellet the cell debris and the nucleic acids are purified from the supernatant.
When the spin column format is employed, several additional centrifugations are performed. Because these methods require at least two centrifugation steps, they are time-consuming, laborious and difficult to fully automate. They require significant human intervention and cannot be performed in a walk-away fashion. Therefore, there exists a need for a more automated methods of plasmid and nucleic acid preparation.
Furthermore, plasmids purified by existing commercially-available methods often contain significant amounts of endotoxin, making them unsuitable for transfection. Therefore, there exists a need for automated, high-throughput nucleic acid purification. Pipette tip columns can be used to meet this need. Additionally, there exists a need for purifying nucleic acids from unclarified cell lysates and other samples containing particulates and cell debris. For plasmid purification, there is a need for endotoxin-free plasmid.
Additionally, there exists a need for large-scale automated nucleic acid preparation. ThermoFisher Scientific offers the BenchPro instrument designed to automatically purify two maxi-scale plasmid preparations. However, the BenchPro instrument can only accommodate up to 125 mL of bacterial culture grown in LB which is a maximum of approximately=1.25×1011 total cells. The maximum yield of plasmid DNA that can be obtained from the BenchPro 2100 is 1.5 mg. The BenchPro cannot accommodate larger volumes or bacterial cultures grown in a rich medium such as Terrific Broth (TB). Therefore, there exists a need for higher yield, larger-scale, automated plasmid preparations. Furthermore, since the BenchPro 2100 is limited to the maxiprep scale, there exists a need for instrument that can perform megaprep and gigaprep plasmid purifications